A unified framework based on the binding polynomial for characterizing biological systems by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has become the gold-standard technique for studying binding processes due to its high precision and sensitivity, as well as its capability for the simultaneous determination of the association equilibrium constant, the binding enthalpy and the binding stoichiometry. The current widespread use of ITC for biological systems has been facilitated by technical advances and the availability of commercial calorimeters. However, the complexity of data analysis for non-standard models is one of the most significant drawbacks in ITC. Many models for studying macromolecular interactions can be found in the literature, but it looks like each biological system requires specific modeling and data analysis approaches. The aim of this article is to solve this lack of unity and provide a unified methodological framework for studying binding interactions by ITC that can be applied to any experimental system. The apparent complexity of this methodology, based on the binding polynomial, is overcome by its easy generalization to complex systems

Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease

Deconvolution analysis for classifying gastric adenocarcinoma patients based on differential scanning calorimetry serum thermograms

Recently, differential scanning calorimetry (DSC) has been acknowledged as a novel tool for diagnosing and monitoring several diseases. This highly sensitive technique has been traditionally used to study thermally induced protein folding/unfolding transitions. In previous research papers, DSC profiles from blood samples of patients were analyzed and they exhibited marked differences in the thermal denaturation profile. Thus, we investigated the use of this novel technology in blood serum samples from 25 healthy subjects and 30 patients with gastric adenocarcinoma (GAC) at different stages of tumor development with a new multiparametric approach. The analysis of the calorimetric profiles of blood serum from GAC patients allowed us to discriminate three stages of cancer development (I to III) from those of healthy individuals. After a multiparametric analysis, a classification of blood serum DSC parameters from patients with GAC is proposed. Certain parameters exhibited significant differences (P < 0.05) and allowed the discrimination of healthy subjects/patients from patients at different tumor stages. The results of this work validate DSC as a novel technique for GAC patient classification and staging, and offer new graphical tools and value ranges for the acquired parameters in order to discriminate healthy from diseased subjects with increased disease burden

Spatial arrangement of LD motif-interacting residues on focal adhesion targeting domain of Focal Adhesion Kinase determine domain-motif interaction affinity and specificity

Background: Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. Methods: Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. Results: This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 µM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 µM vs 63 µM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10-5 µm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10-3 µm2/s). Conclusions: We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. General significance: Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration

W196 and the ß -Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/+) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions - the flexible regulatory C-loop and the 190-202 ß-harpin - promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the ß-hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the ß-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the ß-hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD+. Finally, the ß-hairpin motif contributes to define the conformation of AIF's interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF''s cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNALeu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme–tRNA complex in a novel ‘aminoacylation-like’ conformation, forming novel interactions with the KMSKS loop and the tRNA 30-end. Our findings reveal an intriguing tRNAdependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design

Rescuing compound bioactivity in a secondary cell-based screening by using gamma-cyclodextrin as a molecular carrier

In vitro primary screening for identifying bioactive compounds (inhibitors, activators or pharmacological chaperones) against a protein target results in the discovery of lead com- pounds that must be tested in cell-based efficacy secondary screenings. Very often lead com- pounds do not succeed because of an apparent low potency in cell assays, despite an excellent performance in primary screening. Primary and secondary screenings differ significantly accord- ing to the conditions and challenges the compounds must overcome in order to interact with their intended target. Cellular internalization and intracellular metabolism are some of the difficulties the compounds must confront and different strategies can be envisaged for minimizing that prob- lem. Using a novel screening procedure we have identified 15 compounds inhibiting the hepatitis C NS3 protease in an allosteric fashion. After characterizing biophysically the interaction with the target, some of the compounds were not able to inhibit viral replication in cell assays. In order to overcome this obstacle and potentially improve cellular internalization three of these compounds were complexed with gamma-cyclodextrin. Two of them showed a five- and 16-fold activity increase, compared to their activity when delivered as free compounds in solution (while gamma-cyclodextrin did not show antiviral activity by itself ). The most remarkable result came from a third compound that showed no antiviral activity in cell assays when delivered free in solu- tion, but its gamma-cyclodextrin complex exhibited a 50% effective concentration of 5 micromoles. Thus, the antiviral activity of these compounds can be significantly improved, even completely rescued, using gamma-cyclodextrin as carrier molecule

Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. Databases PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively

Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation